The interplay between environmental exposures, respiratory tract microbiome, and immune responses related to asthma and other respiratory diseases is not well understood. High levels of traffic-related air pollutants (TRAP) have been associated with children's asthma. TRAP can increase adherence of microorganisms to the epithelial cells of the respiratory tract and damage the epithelial layers resulting in increased susceptibility to microbial growth. Many studies suggest a role for altered human microbiota in the etiology of asthma. Furthermore, circumstantial evidence indicates that bacterial infections in the respiratory tract may play a role in asthma development. The airway microbiota may interact with the innate and adaptive arms of the children's developing mucosal immune system in the respiratory tract, which can be critically important in maintaining tolerance against allergc immune responses. Our recent data show that increased exposure to traffic-related particles at birth is associated with longitudinal childhood wheezing. We hypothesize that exposure to TRAP early in life significantly alters the diversity of microorganisms in the respiratory tract in chilren and this effect persists to early adolescence. In Specific Aim 1, we will characterize the respiratory tract microbiome of adolescent children exposed to high and low levels of traffic related air pollution during childhood. Children from the existing cohort of the NIEHS-funded Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS) will be recruited for this purpose. This cohort is well characterized regarding childhood exposure to TRAP and indoor aeroallergens as well respiratory health of children from birth to age 12. TRAP exposure at ages 12-15 will be estimated by a land use regression (LUR) model of exposure to truck and bus traffic. Bacterial composition, operational taxonomic units (OTUs), and diversity indices in the respiratory tract of children will be characterized by collecting induced sputum samples, extracting DNA, amplifying bacteria-specific PCR products (using 16S rRNA primers), analyzing DNA sequences by deep sequencing, clustering and assignment of Illumina MiSeq reads into Operational Taxonomic Units (OTUs), analysis of OTUs, and determination of bacterial diversity by RDP database and pipeline, as well as MG-RAST and Qiime software packages. In Specific Aim 2, we will assess associations between bacterial OTUs, diversity indices, and TRAP. To our knowledge, there are no previous reports on the effects of air pollutants on the human respiratory tract microbiome, particularly among children. This information is critically important to understand the interaction between air pollution, human microbiome, and respiratory health among children.